Protein–Ligand Interaction Detection with a Novel Method of Transient Induced Molecular Electronic Spectroscopy (TIMES): Experimental and Theoretical Studies

Abstract

Protein–ligand interaction detection without disturbances (e.g., surface immobilization, fluorescent labeling, and crystallization) presents a key question in protein chemistry and drug discovery. The emergent technology of transient induced molecular electronic spectroscopy (TIMES), which incorporates a unique design of microfluidic platform and integrated sensing electrodes, is designed to operate in a label-free and immobilization-free manner to provide crucial information for protein–ligand interactions in relevant physiological conditions. Through experiments and theoretical simulations, we demonstrate that the TIMES technique actually detects protein–ligand binding through signals generated by surface electric polarization. The accuracy and sensitivity of experiments were demonstrated by precise measurements of dissociation constant of lysozyme and N-acetyl-d-glucosamine (NAG) ligand and its trimer, NAG3. Computational fluid dynamics (CFD) computation is performed to demonstrate that the surface’s electric polarization signal originates from the induced image charges during the transition state of surface mass transport, which is governed by the overall effects of protein concentration, hydraulic forces, and surface fouling due to protein adsorption. Hybrid atomistic molecular dynamics (MD) simulations and free energy computation show that ligand binding affects lysozyme structure and stability, producing different adsorption orientation and surface polarization to give the characteristic TIMES signals. Although the current work is focused on protein–ligand interactions, the TIMES method is a general technique that can be applied to study signals from reactions between many kinds of molecules.

Nanoscale Ion Emitters in Native Mass Spectrometry for Measuring Ligand–Protein Binding Affinities

“…35 Lysozyme is an antimicrobial enzyme that catalyzes the hydrolysis of β-1,4-glycosidic linkages in specific Gram-positive bacterial walls, 36 and lysozyme–ligand complexes have been studied extensively by native MS 6,7,37,38 and other biophysical chemistry methods. 19 The third protein is a model cytochrome P450 enzyme, CYP199A4 from the bacterium Rhodopseudomonas palustris strain HaA2. 39 Cytochrome P450s are ubiquitous heme-monooxygenases that catalyze the insertion of an oxygen atom from dioxygen into the carbon–hydrogen bonds of organic molecules and other reactions 40 involved in metabolism.…”

“…The interactions between proteins and ligands are crucial to proper cellular function. , The structures, functions, and interactions of protein–ligand complexes can be significantly affected by salts. − Specific metal ion cofactors can regulate the bioactivity of proteins . In native mass spectrometry (MS), ligand–protein interactions are normally stabilized using volatile salts at high ionic strengths to rapidly and directly measure the mass, binding stoichiometry, and relative ligand–protein binding affinities with high sensitivity. − However, most biochemical approaches to probe protein–ligand interactions, including nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, and optical spectroscopy, use nonvolatile salts that can more accurately reflect the in vivo environment of the protein–ligand complex. However, nonvolatile salts and common biological buffers readily adduct to proteins ions to result in broad spectral peaks that have deleterious effects on mass spectra by lowering the sensitivity and signal-to-noise ratios and increasing background chemical noise .…”

Detecting Protein–Ligand Interaction from Integrated Transient Induced Molecular Electronic Signal (i-TIMES)

“…Lysozyme is an antimicrobial enzyme that forms part of the innate immune system. N , N ′, N ″-triacetylchitotriose (TriNAG) is an inhibitor which binds to the active site of lysozyme with a K d of 10–30 μM according to literatures. ,, Figure a and b shows the current signal of lysozyme over a concentration range from 0.3 nM to 200 μM. Figure c shows the i-TIMES signals obtained by integrating the current from Figure a and b.…”

“…Some time ago, we invented the method of transient induced molecular electronic signal (TIMES) to measure protein–ligand reactions in a label-free and immobilization-free manner. , In the TIMES technique, the reaction occurs in solution, and the reaction products are brought to an electrode surface via a microfluidic channel. The molecular constituents in the laminar flow approach the electrode by diffusion and induce changes in the surface charge on the electrode surface, generating a transient current that is amplified by a transimpedance amplifier connected to the electrode.…”

Measuring Electric Charge and Molecular Coverage on Electrode Surface from Transient Induced Molecular Electronic Signal (TIMES)

“…In this paper, we have extended the method of Transient Induced Molecular Electronic Signal (TIMES) reported earlier to directly measure the surface charge for any solution in contact with a conductive surface 24,25 . The results produce the amount and polarity of the surface charge in solution that is in contact with the electrode surface.…”